1. Field of the Invention
This invention relates to reference solutions for use as supplementary components of diagnostic tests performed on serum and other bodily fluids for chronic overconsumption of alcoholic beverages.
2. Description of the Related Art
Diagnostic methods for the detection of alcoholism and alcohol abuse are useful in prescribing treatment to individuals suffering from these conditions and are an important tool in lessening the attendant health complications and the societal consequences that often result. The diagnostic methods consist of determining the level of one or more species in the subject's bodily fluid that serve as biochemical markers for chronic or excessive alcohol consumption. These markers are .gamma.-glutamyltransferasc (GGT), aspartaie aminotransferase (AST), alanine amino transferase (ALT), and carbohydrate-deficient transferring (CDT). Studies have shown that the last of these assays is particularly sensitive and has greater specificity for the condition than the others.
The term "carbohydrate-deficient transferring" or "CDT" refers to certain isoforms of transferring that are formed by conversion of transferring and are present in elevated concentration in the bodily fluids of chronic alcohol abusers. Transferring itself (i.e., normal or intact transferring), which is the iron-transporting protein in blood, is a glycoprotein with two N-linked polysaccharide (or carbohydrate) chains, each of which contains either two or three branches. Each branch is terminated with a sialic (N-acetylneuraminic) acid residue. Transferring exists in a variety of isoforms, differing in the number of polysaccharide chains and the number of branches on each chain. Five such isoforms have been identified by electrochemical separation means based on differences in their pI. The higher the pI, the fewer the polysaccharide chains (and the fewer the sialic acid residues) on the molecule. The major isoform is one that has a pI of 5.4, while those that are elevated in subjects that are chronic alcohol abusers are those having pI's of 5.7 or greater. Thus, the term "carbohydrate-deficient transferrin" or "CDT" refers to transferring isoforms whose pI is 5.7 or greater. (The terms "desialylated transfcrrin" and "dTf" are also used to denote the same isoforms.) The CDT fraction in normal subjects is less than 0.8% of the total transferring, and can rise to as much as ten times that amount in alcohol abusers.
Diagnostic tests for CDT typically begin with the separation of CDT from intact transferring by ion-exchange chromatography. Quantitation of the CDT in the eluate is then achieved by either radioimmunoassay or turbidimetric measurement. Other methods involve the use of high-performance liquid chromatography (HPLC), isoelectric focusing, or immunoblotting in place of ion-exchange chromatography. In each case a reference is needed to check the condition of the assay components, to monitor the precision and accuracy of the method, or to translate the test result to an numerical figure representing the CDT level (i.e., to calibrate the test). To be effective, the reference should be as sensitive as an actual patient sample to the analytical variances that are encountered in the typical clinical laboratory. The reference should also be stable over long periods of time so that it can be stored until ready for use.
The term "reference" is used throughout this specification to denote any fluid composition containing CDT at a known and preselected concentration. Solutions of known concentrations are useful for a variety of purposes. One use is as a control for purposes of determining that a particular test kit or apparatus, or components of the test kit, have not deteriorated during shipping, storage, or handling and arc functioning in the intended manner. Another use is as a standard for calibration or for verification of the linearity of the test response for translating test readings into numerical values for percent CDT. Whether the reference serves as a control or a standard, it is often useful to have two or more such references at different CDT levels. When used as controls, one reference may have a CDT level in the range of a healthy subject and another in a range representing an alcohol abuser. When used as calibrators, multiple references are useful in bracketing the ranges and particularly the threshold value that differentiates normal patients from those suffering from alcohol abuse.
Methods of preparing references exist in the prior art. These involve either screening the plasma or serum from blood donors to identify units with CDT levels in the target ranges, or isolating and separating CDT from units of normal concentration and using the separated CDT to spike a base matrix such as human serum. Both approaches are time-consuming and costly. The former requires extensive testing and may not produce sufficient amounts of the reference for commercial use. The latter entails labor-or capital-intensivc chemical separation techniques including dialysis, precipitation, electrophoresis, and chromatography. As a result, purified CDT is presently available from commercial suppliers only at high cost. The present invention is directed to satisfying the need of supplying CDT reference solutions at low cost for use for any of the purposes described above, with high reproducibility from lot to lot and with ease of manufacturing.